Practical applications of DNA fingerprinting

1. Paternity and Maternity 
Because a person inherits his or her VNTRs from his or her parents, VNTR patterns can be used to establish paternity and maternity. The patterns are so specific that a parental VNTR pattern can be reconstructed even if only the children's VNTR patterns are known (the more children produced, the more reliable the reconstruction). Parent-child VNTR pattern analysis has been used to solve standard father-identification cases as well as more complicated cases of confirming legal nationality and, in instances of adoption, biological parenthood.
2. Criminal Identification and Forensics 
DNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a crime can be compared, through VNTR patterns, with the DNA of a criminal suspect to determine guilt or innocence. VNTR patterns are also useful in establishing the identity of a homicide victim, either from DNA found as evidence or from the body itself.

3. Personal Identification 
The notion of using DNA fingerprints as a sort of genetic bar code to identify individuals has been discussed, but this is not likely to happen anytime in the foreseeable future. The technology required to isolate, keep on file, and then analyze millions of very specified VNTR patterns is both expensive and impractical. Social security numbers, picture ID, and other more mundane methods are much more likely to remain the prevalent ways to establish personal identification.

Meselson and Stalh experiment

Meselson and Stalh used two different isotopes of nitrogen in order to give two different densities to cell growth. . The DNA of the cells grown in ¹⁵N medium had a higher density than cells grown in normal ¹⁴N medium.  After that, E. coli cells with only ¹⁵N in their DNA were transferred to a ¹⁴N medium and were allowed to divide; the progress of cell division was monitored by microscopic cell counts and by colony assay.

DNA was extracted periodically and was compared to pure ¹⁴N DNA and¹⁵N DNA. After one replication, the DNA was found to have intermediate density. Since conservative replication would result in equal amounts of DNA of the higher and lower densities (but no DNA of an intermediate density), conservative replication was excluded. 

Conclusion of experiment

• Results show that after one generation, the double stranded DNA is 1/2 heavy (from the parent) and 1/2 light (newly synthesized). This means that 100% of the strands are of intermediate density.
• After a second generation, one half of the new daughter strands are light (using ¹⁴N DNA as template and synthesizing ¹⁴N NA) and one half are intermediate density (using ¹⁵N DNA as a template and ¹⁴N DNA for synthesis). This result is predicted by semiconservative replication.
• Conclusion- as predicted by Watson and Crick, DNA strands serve as templates for their own replication.

Result of experiment

 After one replication, the DNA was found to have intermediate density. Since conservative replication would result in equal amounts of DNA of the higher and lower densities (but no DNA of an intermediate density), conservative replication was excluded. However, this result was consistent with both semiconservative and dispersive replication. Semiconservative replication would result in double-stranded DNA with one strand of ¹⁵N DNA, and one of ¹⁴N DNA, while dispersive replication would result in double-stranded DNA with both strands having mixtures of ¹⁵N and ¹⁴N DNA, either of which would have appeared as DNA of an intermediate density.

Characteristics of genetic code

Characteristics of genetic code

Following are the characteristics of genetic code accepted world wide

1. Only 61 triplets or codons code for amino acids
   3 stop codons (aka nonsense codons or terminator codons) UUA UAG UGA.
    
2. The code is a degenerative code
   Several codons code for the same amino acid.
   The first two letters seem to be the most important the third one tends to be interchangeable
3. The is no punctuation between each codon.
   The reading frame is set at the beginning of the gene. Frame shift mutations can be caused by the ADDITION or DELETION of only one or two bases. Everything downstream is misread.
    
4. The reading of mRNA is always in the same direction 5’ to 3’ (the same way as transcription and replication).
   The polypeptide chain is constructed from the amino end to the carboxyl end.
    
5. The code is universal for all organisms. So it is very ancient.
    
6. Similar amino acids have similar codons.
   Example
   Aspartic acid codons GAU and GAC. Glutamic acid codons GAA and GAG. Both are acidic amino acids.
    
7. Some amino acids are chemically altered AFTER translation.
   e.g. In collogen proline is converted to hydroxyproline.

Alternative splicing and one gene one polypeptide hypothesis

Alternative splicing is a regulated process during gene expression that results in a single gene coding for multiple proteins. in this process, particular exons of a gene may be included within, or excluded from, the final, processed messenger RNA (mRNA) produced from that gene. Consequently the proteins translated from alternatively spliced mRNAs will contain differences in their amino acid sequence and, often, in their biological functions. Notably, alternative splicing allows the human genome to direct the synthesis of many more proteins than would be expected from its 20,000 protein-coding genes. It has been proposed that for eukaryotes alternative splicing was a very important step towards higher efficiency, because information can be stored much more economically. Several proteins can be encoded by a single gene, rather than requiring a separate gene for each, and thus allowing a more varied proteome from a genome of limited size. So the previous one gene one polypeptide hypothesis in no more valid, now one gene many polypeptide hypothesis is valid. 

Role of Vitamin B6 in Protein Metabolism

Vitamin B complex consist of  water soluble Vitamins which were earlier thought of as being a single Vitamin but later found to be composed of  EIGHT chemically distinct Vitamins. One of these eight vitamins is vitamin B6 or Pyridoxal.

Sources of vitamin B6 in food include

·         Royal Jelly of Bees

·         Yeast

·         Rice Polishing

·         Cereal Grains

·         Egg Yolk

·         Germinal portion of various Seeds

Structure of vitamin B6

Vitamin B6 consists of derivatives of  Purine Ring. This vitamin is Biochemically active only when in Phosphorylated form. This Phosphorylation is brought about by Kinase enzyme that bring about this Phophorylation  at 5^th Position.

Isoforms

Vitamin B6 exists as six isoforms. Which include:

·         Pyridoxine

·         Pyridoxine-P

·         Pyridoxal

·         Pyridoxal-P

·         Pyridoxamine

·         Pyridoxamine-P

·          

The Biologically active forms of vitamin B6 are

a)      Pyridoxal-PO₄

b)      Pyridoxamine-PO₄

Role in amino acid metabolism

Vitamin B₆ is actually the generic name for the precursors of the coenzyme pyridoxal phosphate (PLP). When dietary form of vitamin B6 is taken the phosphate of this coenzyme is removed by intestinal alkaline phosphates, and only the dephosphorylated forms are absorbed. The total body content of PLP is only 25 mg in adults, the circulating form of this vitamin are Pyridoxal and PLP. Several enzymes Involved in amino acid metabolism have PLP as a tightly bound prosthetic group. In these reactions, the aldehyde group of PLP forms an aldimine derivative with the amino group of the amino acid. The aldimine is stabilized by a hydrogen bond with the phenolic hydroxyl group

Pyridoxal phosphate is involved in almost all amino acid metabolisms, from synthesis to breakdown.

Transamination

The Transaminases are the enzymes that break down amino acids. They are dependent on the presence of Pyridoxal phosphate. These enzymes are needed for the process of moving amine groups from one amino acid to another. These Transaminases catalyze the transfer of  NH2 groups from the amino acids, onto alpha-ketoglutarate.  Naturally Many different transaminases are known, and all require the same co-factor  Pyridoxal phosphate (vitamin B6).

Mechanism of Transamination

PLP plays a central role here in the inter conversion of an amino acid and an alpha-keto acid.
(1) Transaminase first bind to pyridoxal phosphate in a Schiff-base link to a Lysine residue of enzyme. This leads to the formation of an "aldimine".
(2) As a new substrate enters the active site, its amino group displaces the -NH₂ of active site Lysine. Then a new Schiff-base link is formed to the alpha-amino group of the substrate, as the active site Lysine moves aside.
(3) There is an electronic rearrangement resulting in shifting the double bond to form a "ketimine".
(4) This formation of ketimine is followed by hydrolysis to release PMP and an alpha-keto acid.
(5) PMP combines with alpha-ketoglutarate in a reversal of steps 1-4. The net result is transfer of an amino group to alpha-ketoglutarate, and release of glutamate, while regenerating the PLP-enzyme complex.

Trans-sulfuration

 Cystathionine synthase and cystathionase need pyridoxal phosphate for proper functioning. These enzymes transform methionine into cysteine. Cystathionine-β-synthase, which is also known as CBS, is an enzyme that is encoded by the CBS gene. It catalyzes the first step of the transsulfuration pathway. CBS also uses the cofactor pyridoxal-phosphate (PLP) 

Selenoamino acid metabolism

 The primary dietary form of selenium is Selenomethionine. For the use of selenium as a nutrient Pyrodixal phosphate act as a co-factor. Pyridoxal phosphate also act as a co-factor in breaking of selenohomocystein to release selenium to produce hydrogen selenide, which can then be used to incorporate selenium into selenoproteins to be used for various cellular functions.

Conversion of tryptophan to niacin vitamin B6

The liver synthesize niacin from tryptophan, liver requires 60 mg of tryptophan to make one mg of niacin. The 5 membered aromaticheterocycle of tryptophan is cleaved and then it is rearranged with the alpha amino group of tryptophan into the 6-membered aromatic heterocycle of niacin. 

Decarboxylation Reactions

Decarboxylation reactions require vitamin b6 as coenzyme for enzymes decarboxylase that is involved in removal of CO₂ to produce AMINES. For example during the conversion of histidine to histamine decarboxylation occurs as removal of CO2 happens and use vitamin b6 as co-enzyme. Other decarboxylation reactions that use vitamin B6 as coenzyme include the conversion of glutamine acid to Gama amino butaric acid (GABA) and conversion of 5-OH Tryptophan to 5-OH Tryptamine (Serotonin) .

Other uses of vitamin B6

1.      Synthesis of sphingosine

2.      Intramitochondrail fatty acid synthesis

3.      Transport of K.

Vitamin B6 deficiency

The deficiency of vitamin B6 occurs rarely and is often associated with malabsorption syndromes and alcoholism. Certain drugs are also thought to cause its deficiency by rendering the vitamin inactive these drugs include isoniazid hydrolazine and penicillamine.

The symptoms of Vitamin B6 deficiency in adults include.

·         Nervousness

·         Irritability

·         Insomnia

·         Muscle weakness

·         Difficulty in walking

·         Glossitis

·         Cheliosis

·         Seizures

Vitamin B₆ deficiency can be prevented or treated with consumption of the recommended dietary allowance, as supplied by food or by vitamin supplements.

Restriction enzymes

Restriction enzymes (endonuleases) are the enzymes that cut DNA at specific sequences. They have the ability to break various kinds of bonds that include

1.      Covalent bonds (within a single strand)

2.      Hydrogen bonds (between strands)

These enzymes are naturally found in different types of bacteria where these Bacteria use restriction enzymes to protect themselves from foreign DNA. Bacteria have mechanisms to protect themselves from the actions of their own restriction enzymes.

Recombinant DNA is constructed using restriction enzymes. Restriction enzymes are used scientifically in various processes e.g.

·         Determination of the size of a plasmid

·         To find out if there are any restriction sites for a particular enzyme on a piece of DNA (ex. EcoRI)

·         To find out how many restriction sites for a particular enzyme. 

·         To locate the restriction sites.

Polymerase  Chain Reaction (PCR)

Polymerase chain reaction enables large amounts of DNA to be produced from very small samples (0.1ml).There is a repeating cycle of separation of double DNA strandsand synthesis of a complementary strand for each.

PCR is a simple process that can be summarized as follows:

The Sample DNA, nucleotides, DNA primers and thermostable DNA polymerase are placed in PCR machine. Then the strands of sample DNA are separated by heating to 95^oC then the mixture is cooled to 37^oC to allow primers to bind. This Mixture is then heated to 72^oC for replication (this is the optimum temp of DNA polymerase). This cycle is repeated many times (~8mins /cycle).

There are certain limitations to the process of PCR for example the separation is achieved by heating to DNA strands to 95^oC while there is no suitable helicase for this purpose. DNA polymerase can’t work on completely single stranded DNA so double stranded regions are needed at the start of sequence to be copied. The primers (short sequences DNA) complementary to bases at start of region to be copied are to be used and to synthesize primers, base sequence at start must be known.

PCR has got a variety of uses some of which are:

·         PCR is used for the detection of infectious agents such as HIV, hepatitis, HPV, EBV, malaria and anthrax
·         PCR is a very value able tool of the diagnosis of certain mutation leading to certain cancers in our body. Is has been used as a prognostic tool for leukemias.
·         PCR is now a day also used to detect genetic abnormalities in post natal or even in in utero periods. Parental testing can also be done using PCR.
 

Plasmids

Plasmid

Plasmids are Extrachromosomal DNA, usually circular in shape. They Usually encode ancillary functions for in vitro growth. Plasmids are required for specific cellular functions like: produnction of virulence, formation of antibiotics resistancea and production of bacteriocins (colicins). For a plasmid to survive it must be a replicon I.e. self-replicating genetic unit. Its DNA must replicate every time host cell divides or it will be lost.

High copy plasmids are usually small and low copy plasmids can be large. Plasmid replication requires host cell functions and host cellular machinery. Copy number is regulated by initiation of plasmid replication. As plasmids interfere with each other’s replication they are incompatible when they cannot be stably maintained in the same cell.

A Plasmid replication requires host DNA replication machinery. Most wild plasmids carry required genes needed for transfer and copy number control. All self replication plasmids have a oriV which is origin of replication gene while some plasmids carry and oriT which origin of transfer gene.  These plasmids will also carry mob genes for mobilization purpose. There are 5 main “incompatibility” groups of plasmid replication.  But not all plasmids can live with each other.

F-plasmid has a large (100 kb) and a low copy (1-2 copies/cell). They are self transmissible and requires protein synthesis. repE gene encodes RepE protein. This RepE protein binds to origin of replication (oriS) and initiates DNA replication. RepE binds to the repE promoter and activates transcription. This RepE binds to the copA/incC locus binding copies of F together via RepE – inhibiting replication (coupling).

Several F plasmids have different functions. For example the plasmids ccdAB is reuired for inhibition of host cell division. incBCE has got incompatibiltity function. onV has a role in bidirectional replication.

Other scientific functions of plasmids include.

1.      Carry antibiotic resistance.

2.      Carry genes for metabolic activity.

3.      They have the ability to produce antibacterial proteins.

4.      They can contain genes for virulence factors for many bacterias.

Pernicious anemia and role of Cobalamine in amino acid metabolism.

Pernicious anemia is a type of megaloblastic anemia that occurs due to abnormal absorption of vitamin B12 (cobalamine) from terminal ileum which is in turn due to decreased production of Intrinsic factor form Parietal cells of gastric mucosa.

This decrease in production of intrinsic factor from gastric parietal cells is due to autoimmune destruction of parietal cells where auto-antibodies are produced against Parietal cells and lead to their destruction and also cause atrophy of gastric mucosa. Genetically people with genotype HLA-DRB*03 and HLA-DRB*04 are more prone to this autoimmune disorder. Some evidence of association of H.pylori infection with pernicious anemia is also seen.

Intrinsic factor

Intrinsic factor is a glycoprotein that is produced form gastric parietal cells. This IF protein is encoded by GIF gene. When we ingest food that contains Vitamin B12, Vitamin B12 is separated from the food by peptic digestion. Gastric parietal cells release haptocorrin that binds to this cobalamine. Now gastric parietal cells release intrinsic factor. This intrinsic factor attaches itself with the cobalamine and separates it from HC. Enterocytes on the other hand have surface receptors for Intrinsic factor. Intrinsic factor attaches itself to those receptors and release cobalamine into the enterocytes. 80% of the cobalamine attaches itself to HC and go towards liver while 20% cobalamine in converted to Holotranscobalamine. The fraction of cobalamine that went to the liver is then release into the duodenum where it is degraded by pancreatic enzymes and HC and cobalamine are separated again. While the fraction of cobalamine that was converted to holotranscobalamine goes to various tissue cells for DNA synthesis.

Cobalamine

Cobalamine or vitamin B12 is a water soluble vitamin that acts in various important body functions. Chemical structure of this vitamin contains cobalt attached to cyanide. Major co-enzyme form of cobalamine is 5’-deoxyadenosyl cobalamine where cobalt-carbon bond is between 5’ carbon of 5’deoxyadenosyl moiety and cobalt of cobalamine.

Vitamin B12 is involved in almost all major metabolic functions of human body. This vitamin cannot be synthesized with the cellular machinery of our cells and thus it has to be taken into the diet. Bacteria have the ability to synthesize this vitamin. The food sources that contain this vitamin include meat, poultry, egg, milk and most important is liver.

Forms of cobalamine

There are various forms of cobalamine present naturally. It depends upon the side molecule to which cobalt is attached.

·         Cyanocobalamine

·         Hydroxycobalamine

·         Adenosylcobalamine

·         Methylcobalamine.

General functions of Cobalamine

Generally cobalamine has a major role in metabolism of fats and proteins. It also acts in the synthesis of folate and methionine. It has a major role in nerve function and production of RBCs. Also it has a role in DNA replication.

Role of cobalamine in amino acid metabolism

When amino acids are deaminated they yield alpha-ketoacid that feeds major metabolic pathways. There are 2 groups of amino acid based on whether or not their carbon skeleton can be converted to glucose.

1.       Glucogenic

2.       Ketogenic

The carbon skeleton of glucogenic amino acids yields pyruvate and 4-C 5-C intermediates of Kreb cycle. While the carbon skeleton of ketogenic amino acids yield acetyl-CoA and acetoacetate.

There are two major roles of Cobalamine in amino acid metabolism

1.       Conversion of homocystein to methionine.

2.       Conversion of propionyl CoA to Succinyl CoA.

Conversion of Homocystein to Methionine

For the sake of conversion of homocystein to methionine. Vitamin B12 is methylated to methylcobalamine by the use of folate. This methylcobalamine is then used to recycle homocystein to methionine. Actually Vitamin b12 act as a co-factor for the enzyme Methionine Synthase that is needed for the conversion of homocystein to methionine.

Conversion of propionyl CoA to Succinyl CoA

For conversion of Propionyl CoA to Succinyl CoA, cobalamine act as a cofactor for enzyme methylmalonyl CoA mutase. This enyme converts Propionyl CoA to Succinyl CoA. Pathway for the conversion of Propionyl CoA to Succinyl CoA is a part of oxidation of various other amino acids like threonine, isoleucine and methionine.

Effects of Vitamin B12 deficiency

In case of vitamin b12 deficiency, homocystein is not converted to methionine and its levels start to increase in blood. The increased levels of homocystein in blood caused endothelial cell damage. This can lead to atherosclerosis and endothelial dysfunction. Some observations also link the elevated blood levels of homocystein with oxidative stress state in our body. Researchers believe it can cause increased risk of CHF. Elevated levels of Homocystein are also associated with Migraine and Stroke.

On the other hand, when Vitamin B12 level decrease in our body, the conversion of Propionyl CoA to succinyl Co A decreases. This leads to decreased oxidation of Amino acids and major metabolic pathways of our body are disturbed. Including the ones associated with Nitrogenous base synthesis. This leads to Immature DNA formation. RBCs with immature DNA continue to grow in size and lead to macrocyte formation. That is why Macrocystic anemia occurs in case of Vitamin B12 deificiency.

Amino acids that are majorly affected by the deficiency of Vitamin B12 are Homocystien, Methionine, Threonine and Isoleucine. Other amino acids are also affected but these are the most effected ones.

Other sign and symptoms of Vitamin B12 deficiency include.

·         Generalized weakness

·         Headache

·         Tachycardia

·         Dyspnea

·         Anemia

·         Neurological manifestations

Essential amino acids

Amino acids are the building blocks of proteins. There are 20 amino acids that make up all of the known proteins in an organism. Chemically amino acids are composed of amino and carboxyl group attached to same alpha-carbon. Generally speaking, amino acids are involved as an intermediate in body’s various metabolic pathways. They also have various catalytic and hormonal functions.

On the basis of our body’s ability to denovo synthesize amine acids, they are divided into two groups:

·         Essential amino acids

·         Non essential amino acids

The amino acids that cannot be synthesized in our body and we require their dietary intake are termed as Essential amino acids. While those who can be denovo synthesized are Non essential amino acids.

Some amino acids are also termed as semi essential, i.e. they cannot be synthesized in the body of children but can be synthesized in adult human body. So children require their dietary intake while adults don’t. These amino acids are Arginine and Histidine.

Essential amino acids are

·         Arginine*

·         Lysine

·         Histidine*

·         Methionine

·         Isoleucine

·         Threonine

·         Leucine

·         Phenylalanine

·         Valine

·         Tryptophan

Among these the starred ones are semi-essential amino acids.

Deficiency of any of the essential amino acid in our dietary intake can lead to various diseases in our body. Structurally, essential and non essential amino acids cannot be distinguished from each other, the only difference is the ability of our body to synthesize non-essential ones and inability to synthesize essential ones.

Arginine

Arginine is a semi-essential amino acid. That means that children don’t have the ability to synthesize arginine but adults usually can synthesize this amino acid. Arginine improves and strengthens the immune system. Foods that contain arginine include flour, almond, walnuts and dairy products. Arginine is also necessary for production of Nitric oxide in endothelial cells. This nitric oxide act as vasodilator and regulator of blood pressure. Some studies also reveal the effects of Arginine on male libido.

Lysine

Lysine is an essential amino acid which our body cannot synthesize on its own. Lysine is a base that is encoded by AAA and AAG codon. Food sources that contain lysine are meat, soy bean, cheese, fish and eggs. Lysine was previously thought to control herpes simplex virus infection but it has not yet been approved by FDA for this purpose. Lysine increases the ability to absorb calcium and decrease the urinary loss of calcium. It also enhances the production of collagen and other connective tissue components.

Histidine

Histidine is a semi –essential amino acid that has negatively charged Imidiazole functional group. Histidine has a major role in myelin sheath formation that insulates the nerve fibers. Histidine also has a major role in production of red blood cells and white blood cells. Studies also reveal the effect of histidine in Platelet formation and function thus effecting blood clotting and homeostasis. Histidine increases the absorption of calcium and decrease histamine levels in our body. Clinically histidine is used during the treatment of rheumatoid arthritis and duodenal ulcers.

Methionine

Methionine is also an essential amino acid. Methionine is encoded by AUG codon. It is a sulphur containing amino acids that act as an intermediate in synthesis of cystein, lectin and phospholipids. Food sources of methionine are Brazil nuts, fish and meat. Major function of methionine is its role as an antioxidant. Its deficiency thus leads to oxidative stress in our body.

Isoleucine

Isoleucine is another essential amino acid that has a branched hydrocarbon chain. Foods that contain this amino acid include eggs, sea weed, chick, lamb and fish. Isoleucine promotes tissue repair and prevent muscle wasting. This amino acid can be converted to sugar in liver.

Threonine

Threonine is an alpha amino acid that is encoded by ACU, ACA, ACC, ACG. Food sources that contain threonine include Cheese, lentils, sesame seeds, meat and fish. Threonine is necessary for biosynthesis of glycine and serine which are needed for connective tissue formation. Threonine improves tissue repair. This amino acid also improves immune system by helping in the biosynthesis of antibodies.

Leucine

Leucine is an essential amino acid that is encoded by UUA, UUG, CUU, CUC, CUA and CUG codons. Leucine is found in various foods that include soy bean, beef, oat and corn. Leucine helps in regulation of blood glucose levels and takes part in tissue repair. Leucine helps in muscle mass building.

Phenylalanine

Phenylalanine is a non polar essential amino acid that is converted to L-tyrosine. L-tyrosine is used in dopamine, norepinephrine and epinephrine biosynthesis. Clinically phenylalanine is used for the treatment of Vitiligo along with UVA exposure. It has also antidepressive action.

Valine

Valine is another essential amino acid that is encoded by GUU, GUC, GUA and GUG. Valine is needed for muscle metabolism and helps maintain nitrogen balance in our body. Valine improves tissue repair, prevents muscle loss and improves muscle coordination.

Tryptophan

Tryptophan is an essential amino acid that is encoded by UGG codon. Food sources that contain this amino acid are Chocolate, peanuts, banana, egg, fish and cheese. Tryptophan has a role in production in Vit B complex (niacin). Tryptophan also acts as an intermediate in serotonin synthesis.

Deficiencies

Deficiency of any of the above mentioned amino acid can lead to several abnormalities in our body’s function that extends from generalized weakness to brain and cardiovascular malfunctions. A balanced and healthy diet should include all of these essential amino acids in required quantities. The amino acids are now also available in form of medications.